Brändli A W, Simons K
European Molecular Biology Laboratory, Heidelberg, FRG.
EMBO J. 1989 Nov;8(11):3207-13. doi: 10.1002/j.1460-2075.1989.tb08479.x.
Sorting of newly synthesized proteins destined for the apical plasma membrane takes place in the trans-Golgi network (TGN) in MDCK cells. This process is most likely receptor mediated and requires components that recycle between both compartments. We have developed an assay to detect apical proteins that recycle through the sialyltransferase-containing TGN. Cell surface glycoproteins were exogalactosylated apically using a mutant cell line derived from MDCK, MDCKII-RCAr. The mutant exhibits impaired galactosylation of glycoconjugates and thereby allows maximal incorporation of exogenously added galactose in the presence of galactosyltransferase. Upon reculture at 37 degrees C, a time-dependent increase of sialylated apical surface glycoproteins was observed by lectin binding as well as by the sialic acid-specific NaIO4/NaB[3H]4 labeling technique. This indicates that some galactosylated surface molecules had returned to the TGN. Recycling through the TGN was blocked, if exogalactosylated cells were incubated at 20 degrees C. Two-dimensional gel electrophoresis identified three apical proteins which recycle through the TGN, suggesting that this pathway is selective for a subset of the apical surface proteins.
在MDCK细胞中，运往顶端质膜的新合成蛋白质的分选发生在反式高尔基体网络（TGN）中。这个过程很可能是受体介导的，并且需要在两个区室之间循环的成分。我们开发了一种检测方法来检测通过含唾液酸转移酶的TGN循环的顶端蛋白质。使用源自MDCK的突变细胞系MDCKII-RCAr对细胞表面糖蛋白进行顶端外半乳糖基化。该突变体表现出糖缀合物半乳糖基化受损，因此在存在半乳糖基转移酶的情况下允许最大程度地掺入外源添加的半乳糖。在37℃再培养时，通过凝集素结合以及通过唾液酸特异性NaIO4/NaB[3H]4标记技术观察到唾液酸化顶端表面糖蛋白的时间依赖性增加。这表明一些半乳糖基化的表面分子已返回TGN。如果将外半乳糖基化细胞在20℃下孵育，则通过TGN的循环被阻断。二维凝胶电泳鉴定出三种通过TGN循环的顶端蛋白质，表明该途径对顶端表面蛋白质的一个子集具有选择性。
